Clinical data from human chronic wounds implicates bacterial biofilm formation with the onset of wound chronicity that leads to prolonged hospitalization and limb amputation. Current approaches for the treatment of wound infections include the application of topical or systemic antibiotic treatments along with wound debridement, drainage, and surgical intervention. However, a critical challenge in treatment of biofilm infection is that they are antibiotic resistant and readily evade innate immune attacks. Thus, there is a dire need for new strategy that targets biofilms in the management of non-healing chronic wounds. To address this challenge, we have recently developed a non-invasive, antimicrobial, magnetic thermotherapy platform in which a high-frequency alternating magnetic field (AMF) is used to rapidly heat magnetic nanoparticles (MNPs) that are bound to a bacterial pathogen. This has been the first application that demonstrates a feasibility of using a targeted MNP hyperthermia as a non-invasive antimicrobial therapeutic for management and accelerated healing of wound infection. Although this technology holds promise, and the use of similar MNPs for hyperthermia has been studied quite extensively in cancer treatment application, there remain several key issues that need to be resolved for the successful clinical translation of this technology to treat wound infections. The goal of the proposed study is to provide preclinical validation of magnetic nanoparticle thermotherapy that cooperates with the innate immune response, works synergistically with conventional antibiotic treatment, is effective in the treatment of polymicrobial biofilm infection with both Gram + and Gram - bacterial species, and ensures safety of the technology. To achieve the goal, we propose (1) to optimize the structure of iron oxide particles conjugates in vitro to effectively target bacterial cells for intracellular heating (Aim 1), (2) to examine the impact of MNP/AMF hyperthermia on innate immune response for bacterial phagocytosis (Aim 2), and (3) to proceed to an in vivo assessment of the MNP/AMF hyperthermia system using clinically relevant diabetic mouse models of cutaneous biofilm infection (Aim 3). The successful completion of this study may provide a needed tool for treatment of fragile patients who cannot tolerate systemic antibiotics and this could establish a novel non-invasive platform for smart treatments of antibiotic-resistant chronic wound infection.